The clinical applications of silver nanoparticles (AgNPs) remain limited due to the lack of well‐established methodologies for studying their nanokinetics. Hereby, the primary goal is to adapt a suite of analytical‐based methodologies for examining the in vitro absorption, distribution, metabolism, and elimination of AgNPs. Vero 76 and HEK 293 cells are exposed to ≈10‐nm spherical AgNPs
+ and AgNPs
? at relevant concentrations (0–300 µg mL
?1) and times (4–48 h). Absorption: Inductively coupled plasma optical emission spectroscopy (ICP‐OES) demonstrates that the two AgNP formulations are not bioequivalent. For example, different bioavailabilities (
C maximum < 20.7 ± 4% and 6.82 ± 0.4%), absorption times (
T maximum > 48 and ≈24 h), and absorption rate laws (first‐ and zeroth‐order at 300 µg mL
?1) are determined in Vero 76 for AgNPs
+ and AgNPs
?, respectively. Distribution: Raman and CytoViva hyperspectral imaging show different cellular localizations for AgNPs
+ and AgNPs
?. Metabolism: Cloud point extraction (CPE)‐tangential flow filtration (TFF) reveal that ≤ 11% ± 4% of the administered, sublethal AgNPs release Ag
+ and contribute to the observed cytotoxicity. Elimination: ICP‐OES‐CPE suggests that AgNPs are cleared via exocytosis.
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